Our long-term goal is to identify molecular pathways that control growth and function of the tunica adventitia. Sonic hedgehog (Shh) is an essential morphogen and growth factor in embryos, is angiogenic in adult tissues, and is required for the formation and maintenance of tissue-specific progenitor cells. Our preliminary data identify a novel Shh signaling domain restricted to the arterial adventitia that supports resident Sca1+ mural (SMC/pericyte) progenitor cells (AdvSca1). Using patched (ptc)-lacZ reporter mice, adventitial Shh signaling was first detected at E15.5-E16.5, was highest from postnatal day 1 (P1) to P10, and colocalized with a sleeve of Shh protein deposited between the media and adventitia. In mice expressing EGFP from the native Shh locus, AdvSca1 cells were strongly EGFPpos while heterogeneous for ptc-lacZ revealing a Shh paracrine signaling network within the adventitia. In Shh-/- mice, AdvSca1 cells were greatly reduced in number suggesting important roles for Shh in recruitment or survival of these progenitor cells. AdvSca1 cells do not express SMC markers in vivo, yet they do express transcription factors required for SMC differentiation such as SRF and myocardin, and readily differentiate to SMC-like cells in vitro. Unlike SMCs, however, AdvSca1 cells express potent co-repressors of SRF-dependent transcription in vivo including Msx1 and KLF4 that may play key roles in maintaining SMC progenitors in the vessel wall. Our overall hypothesis is that Shh signaling in the adventitia is part of a novel signaling network that plays essential roles in vascular development, growth and remodeling, in part, via control of resident AdvSca1 progenitor cells. We propose a working model in which Shh signaling maintains AdvSca1 progenitors in an undifferentiated state via control of potent SRF- dependent transcriptional co-repressors. To test our hypothesis and working model: Specific Aim 1 will localize Shh signaling activity during formation and postnatal growth of the arterial adventitia, and determine which cells in the artery wall produce and respond to Shh in vivo. Specific Aim 2 will employ animal models for vascular wall remodeling together with genetic approaches for gain and loss of Shh signaling to examine functional roles of adventitial Shh signaling in vivo. Specific Aim 3 will determine if AdvSca1 cells are maintained as SMC progenitors by expression of SRF-dependent transcriptional co-repressors, and examine effects of KLF4-deficiency on development of the tunica media, the tunica adventitia and AdvSca1 cells in vivo. PUBLIC HEALTH RELEVANCE: Hedgehog Signaling in the Adventitia Majesky- NIH RO1 application Project Narrative The known risk factors for coronary heart disease (CHD), aneurysm and stroke can account for only about half of the incidence of these diseases in the United States. The research proposed in this application seeks to identify new candidate risk factors for CHD, aneurysm and stroke by studying basic mechanisms for artery wall growth and repair. This work is the first step toward development of new genetic tests for CDH risk, and may lead to identification of new drug targets for more effective treatment of these diseases.